U.S. patent application number 15/650896 was filed with the patent office on 2017-11-02 for optical detection system for flow cytometer, flow cytometer system and methods of use.
The applicant listed for this patent is ACEA Biosciences, Inc.. Invention is credited to Tingting Cheng, Lingbo Kong, Nan Li, Yangde Qing, Xiaobo Wang, Jian Wu.
Application Number | 20170315122 15/650896 |
Document ID | / |
Family ID | 60158899 |
Filed Date | 2017-11-02 |
United States Patent
Application |
20170315122 |
Kind Code |
A1 |
Li; Nan ; et al. |
November 2, 2017 |
OPTICAL DETECTION SYSTEM FOR FLOW CYTOMETER, FLOW CYTOMETER SYSTEM
AND METHODS OF USE
Abstract
An optical engine its use in a bench top flow cytometer, the
optical engine having a set of lasers, each focused horizontally
along an x-axis to a same horizontal position and vertically along
a y-axis to a different vertical position along a same excitation
plane of a flow cell, a set of optics that separate fluorescence of
a same wavelength range into different locations in a focal plane
of collection optics according to the different lasers by which the
fluorescent light is excited; and a detector that selectively
detects light from the different locations thereby distinguishing
between fluorescence emitted within the same wavelength range as
excited by the different lasers.
Inventors: |
Li; Nan; (San Diego, CA)
; Kong; Lingbo; (San Diego, CA) ; Wu; Jian;
(Hangzhou, CN) ; Cheng; Tingting; (Hangzhou,
CN) ; Qing; Yangde; (Hangzhou, CN) ; Wang;
Xiaobo; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ACEA Biosciences, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
60158899 |
Appl. No.: |
15/650896 |
Filed: |
July 15, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15416976 |
Jan 26, 2017 |
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15650896 |
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14547963 |
Nov 19, 2014 |
9575063 |
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15416976 |
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61906367 |
Nov 19, 2013 |
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61994980 |
May 18, 2014 |
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62363032 |
Jul 15, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 15/1459 20130101;
G01N 2015/1402 20130101; G01N 15/1434 20130101; G01N 33/56972
20130101; G01N 2015/1006 20130101; G01N 2015/1477 20130101; G01N
2015/1438 20130101 |
International
Class: |
G01N 33/569 20060101
G01N033/569; G01N 15/14 20060101 G01N015/14; G01N 15/14 20060101
G01N015/14 |
Claims
1. An optical engine for use in a bench top flow cytometer, the
optical engine comprising: a) a set of lasers, each tuned to a
wavelength suited for excitation of fluorescent molecules, wherein
light from each of the lasers is focused horizontally along an
x-axis to a same horizontal position and vertically along a y-axis
to a different vertical position along a same excitation plane,
wherein the same horizontal position along the excitation plane
intersects a flow path through a flow cell of a flow cytometer; b)
a set of optics comprising collection optics for collecting
fluorescence emitted from the flow cell and filtration optics that
filter the emitted fluorescence from the flow cell into different
wavelength ranges, wherein the set of optics further separate the
fluorescence of a same wavelength range into different locations in
a focal plane of the collection optics according to the different
lasers by which the fluorescent light is excited; c) a detector
that selectively detects light from the different locations thereby
distinguishing between fluorescence emitted within the same
wavelength range as excited by different lasers within the set of
lasers and converts light to an electrical signal.
2. The optical engine according to claim 1, wherein the set of
lasers comprises two, three, four or five individual lasers, each
tuned to a different wavelength and directed to a different
vertical position of the excitation plane thereby providing two,
three, four or five distinct vertical positions along the flow cell
respectively.
3. The optical engine according to claim 2, wherein the vertical
positions in the flow cell are separated by between 60 and 120
.mu.m.
4. The optical engine according to claim 3, wherein the vertical
positions in the flow cell are separated by about 80 .mu.m.
5. The optical engine according to claim 1, wherein the collection
optics comprise a half ball lens followed by two sets of doublet
lenses.
6. The optical engine according to claim 1, wherein the filtration
optics comprise a dichroic mirror and/or a bandpass filter.
7. The optical engine according to claim 1, wherein the filtration
optics filter one or more wavelength ranges selected from the group
consisting of 780/60 nm, 615/20 nm, 530/43 nm, 445/45 nm, 586/20,
661/20, 697/58, and 725/40 nm.
8. The optical engine according to claim 1, wherein the different
locations in the focal plane of the collection optics are separated
from adjacent locations by between 1 millimeter to 4 millimeters,
optionally 2 to 3 millimeters.
9. The optical engine according to claim 1, wherein the different
locations in the focal plane of the collection optics are spaced 1
to 4 millimeters apart from adjacent locations within the focal
plane of the collection optics.
10. The optical engine according to claim 1, wherein the set of
optics further comprise a lens for expanding a light beam from each
of the different locations in the focal plane of the collection
optics to a size of about 1 mm to about 3 mm, wherein each light
beam originating from the fluorescence is excited by an individual
laser.
11. The optical engine according to claim 1, wherein the detector
is multi-pixel photon counter (MPPC) or silicon
photomultiplier.
12. The optical engine according to claim 11, wherein the MPPC is
operated with a linear dynamic range above 3 decade.
13. The optical engine according to claim 12, wherein the MPPC is
operated with a linear dynamic range above 4 decade.
14. The optical engine according to claim 11, wherein the MPPC
digital output value is corrected according calibration
factors.
15. The optical engine according to claim 14, wherein the
calibration factors improve linear dynamic range of the MPPC by
more than half decade.
16. The optical engine according to claim 14, wherein the
calibration factors improve linear dynamic range of the MPPC by
more than one decade.
17. The optical engine according to claim 14, wherein the
calibration factors improve linear dynamic range of an MPPC by more
than one and half decade.
18. The optical engine according to claim 14, wherein the
calibration factors improve linear dynamic range of the MPPC by
more than two decades.
19. The optical engine according to claim 1, further comprising a
forward scatter (FSC) detector, a FSC focusing lens, and an
obscuration bar.
20. The optical engine according to claim 19, wherein the
obscuration bar is diamond shaped or has a rectangular shape with
its horizontal dimension being the same as or larger than its
vertical dimension.
21. The optical engine according to claim 19, wherein a perimeter
of the obscuration bar follows a contour of a light intensity
distribution plot, optionally within a 0.1% contour line.
22. The optical engine according to claim 21, wherein the
obscuration bar blocks 99% of unscattered light from detection by
the FSC detector.
23. The optical engine according to claim 1, further comprising a
housing configured to house optical engine components, the optical
engine components comprising the set of lasers, optics for focusing
laser beams to the excitation plane, the collection optics, the
filtration optics, and the detector, wherein a same housing is
configured for interchangeability of different lasers, lenses,
mirrors, filters, and detectors.
24. The optical engine according to claim 1, further comprising the
flow cell.
25. A flow cytometer, comprising: a) the optical engine according
to claim 1; b) a flow cell; and c) a pump in fluid communication
with an aspiration needle for aspirating and delivering a
suspension of cells through the flow cell.
26. The flow cytometer according to claim 25, further characterized
in that a) the set of lasers comprises two, three, four or five
lasers, each laser tuned to a different wavelength and focused to a
different vertical position along the flow cell; and b) the set of
optics spatially distinguishing the filtered fluorescence in the
same wavelength range that is excited by each of the two, three,
four or five different lasers respectively.
27. A flow cytometry system comprising: a) the flow cytometer
according to claim 25; and b) a computer operably loaded with
developed flow cytometry software to acquire and analyze flow
cytometry data.
28. The flow cytometry system according to claim 27, wherein the
software provides programming to perform the following functions:
a) acquiring data of fluorescence channels from each detector,
wherein the fluorescence signals collected by different detectors
are converted to different data series, corresponding to the
fluorescence excited by lasers at the different vertical positions;
b) generating a graphical user interface (GUI) that displays
various plots for the acquired data, wherein the GUI further
comprises compensation scroll bars adjacent to the comparison plots
to adjust compensation of spectral overlap between one or more
channel; and c) saving the acquired data into a data file.
29. A flow cytometry method comprising: a) providing the flow
cytometry system according to claim 27; b) labeling a suspension of
cells with a plurality of fluorescent labels; c) pumping the
suspension of cells through the flow channel; d) collecting flow
cytometry data; and e) analyzing the flow cytometry data to
determine the presence, absence or abundance of one or more of the
plurality of fluorescent labels on or in cells of the sample.
30. An optical engine for use in a bench top flow cytometer, the
optical engine comprising: a) a laser, tuned to a wavelength suited
for excitation of fluorescent molecules, wherein light from the
laser is focused horizontally along an x-axis to a horizontal
position and vertically along a y-axis to a vertical position along
an excitation plane, wherein the horizontal position along the
excitation plane intersects a flow path through a flow cell of a
flow cytometer; b) a set of optics comprising collection optics for
collecting fluorescence emitted from the flow cell and filtration
optics that filter the collected fluorescence from the flow cell
into different wavelength ranges, thereby providing different
fluorescent channels; and c) a MPPC detector at each fluorescent
channel to detect fluorescence and convert light to an electrical
signal.
31. The optical engine according to claim 30, further comprising at
least 1 to 4 additional lasers, wherein each of the lasers is
focused vertically along the y-axis to a different vertical
position along the same excitation plane, further wherein the set
of optics separate the emitted fluorescence from the flow cell into
different fluorescence channels, wherein each channel is
characterized by a different wavelength range and a different laser
by which the respective fluorescence is excited.
32. The optical engine according to claim 30, wherein the MPPC is
operated with a linear dynamic range above 3 decade.
33. The optical engine according to claim 32, wherein the MPPC is
operated with a linear dynamic range above 4 decade.
34. The optical engine according to claim 30, wherein the MPPC
digital output value is corrected according calibration
factors.
35. The optical engine according to claim 34, wherein the
calibration factors improve linear dynamic range of the MPPC by
more than half decade.
36. The optical engine according to claim 35, wherein the
calibration factors improve linear dynamic range of the MPPC by
more than one decade.
37. The optical engine according to claim 36, wherein the
calibration factors improve linear dynamic range of the MPPC by
more than one and one-half decade.
38. The optical engine according to claim 37, wherein the
calibration factors improve linear dynamic range of the MPPC by
more than two decades.
39. A flow cytometer, comprising: a) the optical engine according
to claim 30; b) a flow cell; and c) a pump in fluid communication
with an aspiration needle for aspirating and delivering a
suspension of cells through the flow cell.
40. The flow cytometer according to claim 39, further characterized
in that a) the set of lasers comprises two, three, four or five
lasers, each laser tuned to a different wavelength and focused to a
different vertical position along the flow cell; and b) the set of
optics spatially distinguish the filtered fluorescence in the same
wavelength range that is excited by each of the two, three, four or
five different lasers.
41. A flow cytometry system comprising: c) the flow cytometer
according to claim 39; and d) a computer operably loaded with
developed flow cytometry software to acquire and analyze flow
cytometry data.
42. The flow cytometry system according to claim 41, wherein the
software provides programming to perform the following functions:
a) acquiring data of fluorescence channels from each detector,
wherein fluorescence signals collected by different detectors are
converted to different data series, corresponding to the
fluorescence excited by lasers at the different vertical positions;
b) generating a graphical user interface (GUI) that displays
various plots for the acquired data, wherein the GUI further
comprises compensation scroll bars adjacent to the comparison plots
to adjust compensation of spectral overlap between one or more
channel; c) saving the acquired data into a data file.
43. A flow cytometry method comprising: a) providing the flow
cytometry system according to claim 40; b) labeling a suspension of
cells with a plurality of fluorescent labels; c) pumping the
suspension of cells through the flow channel; d) collecting flow
cytometry data; and e) analyzing the flow cytometry data to
determine the presence, absence or abundance of one or more of the
plurality of fluorescent labels on or in cells of the sample.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation in part of U.S. patent application
Ser. No. 15/416,976, filed Jan. 26, 2017, which is a divisional of
U.S. patent application Ser. No. 14/547,963, filed Nov. 19, 2014,
now U.S. Pat. No. 9,575,063, which claims benefit of priority to
U.S. provisional patent application Ser. No. 61/906,367, filed Nov.
19, 2013 and U.S. provisional patent application Ser. No.
61/994,980, filed May 18, 2014. Each patent application referenced
in this paragraph is herein incorporated by reference in its
entirety.
[0002] This application also claims benefit of priority to U.S.
provisional patent application Ser. No. 62/363,032, filed Jul. 15,
2016; the entirety of which is herein incorporated by
reference.
TECHNICAL FIELD
[0003] The invention relates to flow cytometry instrumentation and
more specifically to an optical detection system that collects
light including fluorescence from different vertical positions in a
flow channel and separates collected fluorescence into a plurality
of different detection channels according to wavelength range and
by vertical position for selective detection.
BACKGROUND OF THE INVENTION
[0004] Flow cytometry is a laser-based, biophysical technology
where fluorescent molecules coupled to cells are passed through a
flow cell and excited by a set of lasers. The fluorescence is
collected and separated into different channels with specific
detection wavelengths, converted to electrical signals, and
analyzed using a computer. By labeling cells with different
fluorophores, various distinct cell populations can be resolved.
For example, multi-color flow cytometry, such as three color flow
cytometry uses fluorophores with different excitation and/or
emission wavelengths to differentiate various cell subpopulations
within biological samples.
[0005] Operationally, an excitation light is delivered to a flow
cell by beam-shaping, steering, and guiding optical components.
Passing fluorescently labeled cells or particles through the flow
cell diffracts the light and excites the labels causing
fluorescence. A complex design of multiple-lenses positioned at
accurate locations relative to each other and relative to flow cell
are employed to collect the fluorescent light and the diffraction
light from the particles. The collected light is then split into
different channels according to the particular excitation lasers
and according to the light wavelength.
[0006] In one approach, different fiber optic cables are used to
collect the fluorescent/scattered light as excited from different
laser sources. Then the light from each fiber optical cable is
split into different fluorescent channels. Alternatively, a
specially designed objective is used to collect light from
particles as they pass through different laser sources and the
light is separated into different beams according to which laser
source the light was generated and separated into different
channels according to different dichroic mirrors.
[0007] All such collection optics are expensive to make, difficult
to align, and difficult to adjust. Also, for many situations, the
light collection efficiency is limited. Furthermore, the collected,
split light is conventionally detected and measured with
photo-multiplier tubes (PMTs). Whilst PMTs are widely used for flow
cytometry applications and other optical measurement situations,
they are expensive, bulky in size and complex to use. Therefore,
there is a need for improved collection optics that are simple in
design, have fewer optic components, have high light-collection
efficiency, and have light detection/measurement
sensitivity/efficiency.
SUMMARY OF THE INVENTION
[0008] The above deficiencies in flow cytometry design and
technical approach are addressed by the present invention. In one
aspect of the invention, an optical engine for use in a flow
cytometer is provided, the optical engine including: a set of
lasers, each tuned to a wavelength suited for excitation of
fluorescent molecules, wherein light from each of the lasers is
focused horizontally along an x-axis to a same horizontal position
and vertically along a y-axis to a different vertical position
along a same excitation plane, wherein the same horizontal position
along the excitation plane intersects a flow path through a flow
cell of a flow cytometer; a set of optics including collection
optics for collecting fluorescence emitted from the flow cell and
filtration optics that filter collected fluorescence from the flow
cell into different wavelength ranges, wherein the set of optics
further separate the fluorescence of a same wavelength range into
different locations in a focal plane of the collection optics
according to the different lasers by which the fluorescent light is
excited; and a detector that selectively detects light from the
different locations thereby distinguishing between fluorescence
emitted within the same wavelength range as excited by different
lasers within the set of lasers and converts light to an electrical
signal. For the present application, light propagation direction
for each laser is defined as Z-axis, which is perpendicular to the
horizontal x-axis and to the vertical y-axis.
[0009] The optical engine permits the use of any number of lasers,
but in some embodiments has at least two lasers, for example, two,
three, four or five lasers, each of which is tuned to a different
wavelength. In preferred embodiments, all the lasers are focused
vertically along the vertical direction to different vertical
positions of the flow cell. In one embodiment, the optical engine
comprises a number of lasers, each emitting light at a specific
wavelength suited for excitation of fluorescent molecules; a set of
beam shaping optics for each laser, wherein each set comprises two
lenses to adjustably focus light horizontally along an x-axis to a
same horizontal position and vertically along a y-axis to a
different vertical position along a same excitation plane, wherein
the horizontal position on the excitation plane interests a flow
path through a flow cell of the flow cytometer. In a preferred
embodiment, a set of beam shaping optics comprises a set of
cylindrical lenses (e.g., an x-axis cylindrical lens and a y-axis
cylindrical lens) or a set of a Powell lenses (e.g. an x-axis
Powell lens and an y-axis Powell lens). In another embodiment, the
optical engine comprises a number of lasers, each emitting light at
a specific wavelength suited for the excitation of fluorescent
molecules; all the lasers' beams being independently adjustable
horizontally along an x-axis and independently adjustable
vertically along a y-axis, being combined together via suitably
placed dichroic mirrors and going through a single achromatic beam
shaping optic so that all the laser beams are focused to a same
horizontal position and to different vertical positions along a
same excitation plane, wherein the horizontal position on the
excitation plane intersects a flow path through a flow cell of the
flow cytometer. This embodiment is different from the example
described where each laser has its own beam shaping optics.
[0010] In still other embodiments, the optical engine comprises a
number of lasers, each emitting light at a specific wavelength
suited for the excitation of fluorescent molecules. Optical design
approaches different from above mentioned two embodiments are
employed so that all the laser beams are focused to a same
horizontal position and to different vertical positions along a
same excitation plane, wherein the horizontal position on the
excitation plane interests a flow path through a flow cell of the
flow cytometer.
[0011] Focused laser beams at the excitation plane shall have beam
sizes suitable for flow cytometry application. Generally, the
vertical beam width at the excitation plane may vary from about two
microns to about 20 microns. In one embodiment, the vertical beam
width is between 2 and 5 microns. In another embodiment, the
vertical beam width is between 5 and 20 microns. Preferably, the
vertical beam width is between 5 and 15 microns. Generally, the
horizontal beam width may vary from as about twenty microns to
about 200 microns. In one embodiment, the horizontal beam width is
between 20 and 50 microns. In another embodiment, the horizontal
beam width is between 50 and 200 microns. Preferably, the
horizontal beam width is between 50 and 100 microns.
[0012] Vertically focusing each of the multiple lasers (e.g., 2
lasers, 3 lasers, 4 lasers, 5 lasers or more) individually at
different vertical positions along a flowing direction of the
sample allows for distinguishing fluorescence excited by each of
the multiple lasers by different photodetectors, as the spatial
separation of the three different lasers along the vertical axis
translates to time and positional differences of fluorescence
emitted by particles when passing through each of the different
lasers. Specially designed collection optics not only collect light
from different vertical locations of the flow cell but also permit
further separation of the light from different vertical locations
as the light propagates through the filtration optics. The
filtration optics, having optical components such as dichroic
mirrors, band pass filters and/or other types of filters or lenses,
can filter the fluorescence and light from the flow cell, into
different wavelength ranges. Thus, light at each of these
wavelength ranges is separated spatially along the vertical axis at
a focal plane of the collection optics, thereby permitting
fluorescence components within a same wavelength range to be
distinguished during detection according to its originating laser.
In some embodiments, the vertical separation between neighboring
vertical positions of the focused beam along the excitation plane
in the flow cells is between 60 and 200 .mu.m. In other
embodiments, the vertical separation between neighboring vertical
positions of the focused beam in the flow cells is between 60 and
100 .mu.m. In still another embodiment, the vertical separation
between neighboring vertical positions of the focused beam in the
flow cells is about 80 .mu.m. The collection optics are able to
amplify such separation distance to achieve a spatial separation of
about a couple mm (e.g. a value between 1.5 and 2.5 mm), or about a
few millimeters (such as about 3 mm, about 3.5 mm, about 4 mm or
about 5 mm) between the neighboring vertical positions at the focal
plane of the collection optics (each vertical position here
corresponds to a light beam of particle fluoresce as excited by one
corresponding laser). Spatial separation of adjacent beams at the
focal plane of the collection optics permits fluorescent signal to
be distinguished by wavelength range and originating laser using
optical detectors.
[0013] In some embodiments, optical detectors are placed at the
corresponding vertical positions along the focal plane of the
collection optics, where each detector detects a light beam of
particle fluoresce as excited by one corresponding laser. Such
optical detectors can be arranged in a form of a detector array. In
other embodiments, optical detectors are placed at some distances
away from the focal plane of the collection optics, wherein each
detector detects a light beam of particle fluoresce as excited by
one corresponding laser. In still other embodiments, optical
detectors are placed at some distances away from the focal plane of
the collection optics and a lens is positioned along the optical
path between the focal plane and the optical detector, wherein each
detector detects a light beam of particle fluorescence as excited
by one corresponding laser. Such a lens could serve the purpose of
expanding the light beam from the focal plane and providing a
relatively-uniform beam distribution.
[0014] In a preferred embodiment, the collection optics include a
half ball lens followed by two sets of doublet lenses. Preferably,
the half-ball lens is made of materials having a high refractive
index. Preferably the combination of two sets of doublet lenses
allow not only collection of light from different vertical
positions in the flow cell but also further focus such light to a
focal plane having larger separation distances of mm range, after
light travels through filtration optics. The filtration optics can
include long pass and/or short pass dichroic mirrors, bandpass
filters, and other filters and/or lenses. In some embodiments, the
filtration optics filter the collected fluorescence light (e.g.
using a half ball lens and two sets of doublet lenses) into
different wavelength ranges characterized as the following
wavelengths 780/60 nm, 615/24 nm, 530/30 nm (or 530/43 nm), 445/45
nm, 586/20 nm (or 572/28 nm), 661/20, 697/58 nm (or 695/40 nm), and
725/40 nm. Note that all the wavelengths have a unit of nm. The
channel wavelengths cited here are for exemplary purposes only and
are not intended for limiting the present invention.
[0015] Various methods can be used to distinguish light spots with
mm-range separation at a focal plane of the collection optics. In
one embodiment, such light spots are separated and focused to
smaller sizes then coupled into a bundle of fiber optic cables. The
light at the end of the fiber optic cables can be detected by a
light detector such as a Photon Multiplier Tube (PMT), a silicon
multiplier or multi-pixel photon counter (MPPC), or a photodiode.
In another embodiment, such light spots at a focal plane of the
collection optics are directly detected by a linear MPPC array,
which comprises multiple MPPC chips, where each chip detects a
corresponding light spot. In yet another embodiment, such light
spots are further separated with additional optical components to
even larger spatial distances between neighboring spots, to be
detected or measured by a number of photo detectors such as a
number of MPPC detectors, or a number of photodiodes, or a number
of avalanche photodiodes, or a number of PMTs. In an exemplary
embodiment, 4 lasers are employed as excitation sources with the
vertical separation of 80 microns between neighboring vertically
focused beams in the flow cell. The vertical separation distance
between neighboring light spots at a focal plane of the collection
optics is about a couple of mm (e.g. 1.5-2.5 mm) or a few
millimeters (e.g., 3-5 mm). The two middle light spots are then
further separated through a prism mirror, each to be detected by a
MPPC detector. In particular, the two side light spots are directly
detected by two MPPC detectors mounted at corresponding
positions.
[0016] In other embodiments of the optical engine of the present
invention, other approaches, different from the collection optics
and filtration optics described above, could also be employed to
collect, separate and split the fluorescent light and the scattered
light from the particles flowing through the flow cell. In one
embodiment, excitation laser beams of different wavelengths are
delivered and focused to a flow cell by beam-shaping, steering, and
guiding optical components. All the focused laser beams share a
common horizontal position and would have different vertical
positions in the flow cell where the flow channel is placed along a
vertical direction. Passing fluorescently labeled cells or
particles through the flow cell diffracts the light and excites the
labels causing fluorescence. A complex design of multiple-lenses
positioned at accurate locations relative to each other and
relative to flow cell are employed to collect the fluorescent light
and the diffraction light from the particles. The collected light
is then split into different channels according to the particular
excitation lasers and according to the light wavelength.
[0017] In one approach of light collection and separation,
different fiber optics cables are used to collect the
fluorescent/scattered light as excited from different laser
sources. Then the light from each fiber optical cable is split into
different fluorescent channels via use of different dichroic
mirrors and bandpass filters. In another approach, a specially
designed objective is used to collect light from particles as they
pass through different laser sources and the light is separated
into different beams according to which laser source the light was
generated. Each separated light beam, originating from one laser
source, is then separated into different channels according to the
use of different dichroic mirrors and bandpass filters.
[0018] A detector for light detection (scattering light or
fluorescent light) in the present invention is provided for each
fluorescence channel, which is preferably in the form of a MPPC
detector. Preferably, fluorescent light signal is converted to an
analog electrical current signal by a MPPC, which is then converted
to an analog electrical voltage signal through the use of a
resistor. Still preferably, analog voltage signals are then
converted to digital signals using analog to digital converter
(ADC) and processed in digital form for increased accuracy and
speed. In a preferred embodiment, each digital output data from the
ADC is corrected or calibrated by dividing the data by a
corresponding calibration factor, determined using the techniques
described in the specification sections below. Preferably, the
calibration factors allow the improvement of linear dynamic range
by at least about half (0.5) decade. More preferably, the
calibration factors allow the improvement of the linear dynamic
range by at least about one (1) decade. Even more preferably, the
calibration factors allow the improvement of the linear dynamic
range by at least about one-and-half (1.5) decade. Even more
preferably, the calibration factors allow the improvement of the
linear dynamic range by at least about two (2) decades.
[0019] In another embodiment of the present invention, an optical
engine for use in a bench top flow cytometer is provided, which
comprises, a laser, tuned to a wavelength suited for excitation of
fluorescent molecules, wherein light from the laser is focused
horizontally along an x-axis to a horizontal position and
vertically along a y-axis to a vertical position along an
excitation plane, wherein the horizontal position along the
excitation plane intersects a flow path through a flow cell of a
flow cytometer; a set of optics comprising collection optics for
collecting fluorescence emitted from the flow cell and filtration
optics that filter the collected fluorescence from the flow cell
into different wavelength ranges, thereby providing different
fluorescent channels; and an MPPC detector at each fluorescent
channel to detect fluorescence and convert light to an electrical
signal. In a preferred embodiment, the optical engine further
comprises a set of lasers, wherein each of the lasers is focused
vertically along the y-axis to a different vertical position along
the same excitation plane, further wherein the set of optics
separate the emitted fluorescence from the flow cell into different
fluorescence channels, wherein each channel is characterized by a
different wavelength range and a different laser by which the
fluorescence is excited.
[0020] In preferred embodiments of above optical engines, the MPPC
is operated with a linear dynamic range above 3 decade. More
preferably, the MPPC is operated with a linear dynamic range above
4 decade.
[0021] In some embodiments of above optical engines, the MPPC
digital output value is corrected according calibration factors.
Preferably, the calibration factors improve linear dynamic range of
the MPPC by more than half decade. More preferably, the calibration
factors improve linear dynamic range of the MPPC by more than one
decade. Even more preferably, the calibration factors improve
linear dynamic range of the MPPC by more than one and one-half
decade. Still, even more preferably, the calibration factors
improve linear dynamic range of the MPPC by more than two
decades.
[0022] In a preferred embodiment, forward scatter (FSC)
characterization of cells includes a FSC detector, a FSC focusing
lens to collect FSC light, and an obscuration bar that blocks an
incident laser beam from entering the FSC focusing lens and the FSC
detector. The relationship between timing of fluorescence signal at
a fluorescent light detector and timing of forward scatter signal
at a FSC detector provides an approach for determining which laser
induces excitation of a detected fluorescent signal in a detection
channel.
[0023] Further improvement of forward scatter (FSC) detection has
been achieved through the use of improved obscuration bars. In a
preferred embodiment, a diamond shaped obscuration bar is provided.
In another embodiment an obscuration bar that is of a rectangular
shape and has its horizontal dimension being the same as or longer
than its vertical dimension is provided for blocking the incident
laser beam. In still another embodiment, the perimeter of the
obscuration bar follows a contour of a light intensity distribution
plot for blocking incident laser beam. In a still further
embodiment, the obscuration bar follows a contour of a light
intensity distribution plot within the 0.1% contour line. A 0.1%
contour line or boundary corresponds to a line where the light
intensity at each point on the contour is at 0.1% of maximum light
intensity of the incident light. An obscuration bar following the
contour of a light intensity distribution plot within the 0.1%
contour was determined to block 99% of the unscattered beam from
the FSC detector. Accordingly, the invention also provides an
obscuration bar generally diamond shaped that follows a contour of
a light intensity distribution plot within the 0.1%, 0.2%. 0.5%,
1.0% or 2.0% contour line and methods of its shaping.
[0024] Components of the optical engine are preferably housed as a
single unit, and some of these optical components can be removed
and interchanged for modification with other components. To this
end, a housing configured to house optical engine components is
also provided. The housing includes the optical engine components
such as the set of lasers, the optics for focusing laser beams to
the excitation plane, collection optics, filtration optics,
photo-detectors or light-detectors, further filters (and/or
lenses), as well as an electrical interface for electrical
connection from the photo-detectors or light-detectors to
electrical circuitry, which would be connected to an external
microprocessor or a remote computer. In some embodiments, each
laser has a corresponding set of beam-shaping optics wherein light
from each laser is focused horizontally along an x-axis to a same
horizontal position and vertically along a y-axis to a different
vertical position along a same excitation plane, wherein the same
horizontal position along the excitation plane intersects a flow
path through a flow cell of a flow cytometer. In other embodiments,
all the laser beams being independently adjustable horizontally
along an x-axis and independently adjustable vertically along a
y-axis, are combined together via suitably placed dichroic mirrors
and go through a common achromatic beam shaping optics so that all
the laser beams are focused to a same horizontal position and to
different vertical positions along a same excitation plane, wherein
the horizontal position on the excitation plane interests a flow
path through a flow cell of the flow cytometer. Preferably, the
components within a same housing are configured for
interchangeability of different lasers, focusing lenses, long pass
and short pass dichroic mirrors, filters, pinhole passages and
detectors. This is accomplished by standardizing engagement
features such as positioning of alignment holes, snaps, screws or
other fasteners across different components for interchangeability
and by providing a set of beam shaping optics for each laser
individually. Preferably, the photo-detectors or the light
detectors are MPPC detectors. In some embodiments, a flow channel
is mounted in the housing and configured for coupling to a flow
cytometer apparatus for hydrodynamic focusing of samples including
particles (e.g. beads or cells) by tubing connectors.
[0025] In a related embodiment, the invention also includes a flow
cytometer, which includes any of the optical engines as disclosed
herein; a flow channel; and a pump in fluid communication with an
aspiration needle for aspirating and delivering a suspension of
cells through the flow channel. In a preferred embodiment, the flow
cytometer is further characterized in that there are two (2), or
three (3), or four (4) or five (5) lasers, each tuned to a
different wavelength and focused to a different vertical position
of the flow cell; a set of optics including collection and
filtration optics for collecting and filtering light from the flow
cell; and where the set of optics spatially distinguish and
separate the filtered fluorescence in the same wavelength range,
that is excited by each of the two, three, four or five different
lasers, to different vertical locations along a focal plane of the
collection optics.
[0026] In a preferred embodiment, the flow cytometer is further
characterized in that there are four lasers, each tuned to a
different wavelength and focused to a different vertical position
of the flow cell (i.e., total four vertical positions in the flow
cell); collection optics for collecting light from the flow cell
and filtration optics for filtering the light; and wherein the
collection optics and filtration optics spatially distinguish
and/or separate the filtered fluorescence based on the vertical
position of the focused excitation beam to different vertical
locations in a focal plane of the collection optics (i.e. also four
distinct vertical positions in the focal plane). Light spots at
such a plane would be, optionally further separated, and detected
by a number of MPPC detectors, or a MPPC array. To this end, a flow
cytometry apparatus is provided which includes up to 25 fluorescent
color channels for particles or cells passing through the flow cell
in addition to side scatter and forward scatter measurement.
[0027] In a related embodiment a flow cytometry system has been
developed, which includes a flow cytometer as provided herein; and
a software for loading and execution in a computer to acquire and
analyze flow cytometry data. As such, flow cytometry software for
loading in a computer has also been developed. In some embodiments,
the software provides programming to perform the following
functions: collecting data from fluorescence channels for each
detector, wherein the fluorescence signals collected by different
detectors are converted to different data series, corresponding to
the fluorescence excited by lasers at the different vertical
positions; generating a graphical user interface (GUI) that
displays various plots for the acquired data, wherein the GUI
further comprises compensation scroll bars adjacent to the
comparison plots to adjust compensation of spectral overlap between
one or more channel; acquiring the data from the cytometer and
saving the data as a data file into the computer hard drive. The
software also includes a gating function that permits the user to
select a subpopulation from a data plot and generate additional
plots for the selected subpopulation. This process can be performed
repetitively for all fluorescence channel data as well as side
scatter and forward scatter data.
[0028] In still another related embodiment a flow cytometry method
is provided, which includes providing flow cytometry system as
provided herein; labeling a suspension of cells with a plurality of
fluorescent labels; pumping the sample of cells through the flow
cell; collecting flow cytometry data; and analyzing the flow
cytometry data to determine the presence, absence or abundance of
one or more of the plurality of fluorescent labels on or in cells
of the sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a schematic providing an overview of transfer of a
cell suspension through the optical engine 100.
[0030] FIG. 2 is a top view of a representation showing an
exemplary optical engine 100.
[0031] FIG. 3 is a schematic top view of showing laser light
propagation along three light paths P1-3 along the Z-axis; wherein
horizontal X-axis is normal to the direction of laser light
propagation (i.e. Z-axis) of an optical illumination system
including 3-laser excitation sources. Also shown is the blocking of
unscattered light from path P2 by the obscuration bar 180.
[0032] FIG. 4 is schematic depicting an enlarged view of the flow
cell 130 showing a common horizontal focus position H for the three
light paths P1-P3 and the different vertical focusing positions Vv,
Vb, Vr of each path P1, P2, P3.
[0033] FIG. 5 is a schematic showing the splitting of collected
light from a flow channel in a flow cell into six different
fluorescent wavelength ranges plus one side scatter channel (SSC).
Using apparatus and methods in the present invention, the six
fluorescent wavelength ranges could correspond to 13 fluorescent
color channels.
[0034] FIG. 6A is a schematic showing fluorescent light from four
different vertical positions (Vv, Vr, Vg, Vb) from a same flow cell
130 collected by collection optics 152, 154, traveling through
light splitting module 142, filtered by a band-pass filter 144,
focused at focal plane 200 of collection optics 152, 154, and
detected within detection module 161 having detectors 162-165.
[0035] FIG. 6B is a schematic showing a configuration for detecting
four fluorescent light beams broadened by a lens 192 (193, 194 or
195) for detection at detector 162 (163, 164 or 165).
[0036] FIG. 7A shows the dependency of the digital output from an
MPPC detector of 1.5 mm.times.1.5 mm in size on the power level of
the incident light. FIG. 7B shows that the dependency of the
digital output from another MPPC detector with different resistors
from that used in FIG. 7A (the resistors are used for converting
MPPC output electric current to electrical voltage). FIG. 7C shows
a linear regression fit of MPPC digital output versus incident
light intensity for the beam size of 1.1 mm in FIG. 7B.
[0037] FIG. 8 shows the dependency of the digital output after
converting electronic analog voltage signals from an MPPC on the
power level of the incident light. The MPPC is of 3 mm.times.3 mm
in size.
[0038] FIG. 9 shows the plot of calibration factor versus MPPC
digital output, as determined for an MPPC having size of 3
mm.times.3 mm in size, at a particular operational bias voltage and
room temperature, for incident light beam of wavelength range
between 515-545 nm.
[0039] FIG. 10 shows that the histogram of dark count noises with
MPPC blocked from any external light with left panel at a room
temperature and the right panel at a lowered temperature.
[0040] FIG. 11 shows a preliminary data of 6-pk beads detected at
the fluorescent channel of 530/43 nm, excite by a blue laser, by a
MPPC detector, with left panel at a room temperature and the right
panel at a lowered temperature.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0041] The invention provides a flow cytometer and its optical
engine that is individually configurable and expandable by
interchangeable lasers, optics configurations and detectors that
provide measurement of up to many parameters, including a large
number of color fluorescence channels, of many individual particles
in a single sample. The interchangeability of the components within
the optical engine permits the user to tailor the excitation and
detection channels according to unique experimental conditions and
according to individual needs. This allows the user to add or
substitute components within a same flow cytometer while
maintaining high detection sensitivity and resolution. The improved
detection sensitivity and resolution is further made possible by
incorporating a multi-pixel photon counter (MPPC) that has high
photon-electron conversion efficiency, yet overcomes the
shortcoming of MPPC devices, namely, larger dark-count and larger
background noises and narrow dynamic ranges.
[0042] The flow cytometer, includes an optical engine, which is
described in various nonlimiting embodiments herein; a flow
channel; and a pump in fluid communication with an aspiration
needle for aspirating and delivering a suspension of cells through
the flow channel. The pump fluidics are shown to reproducibly
deliver cells through the flow channel at high speed to
reproducibly conduct sample acquisition rates of over many
thousands events/second. Further, with the add-on autosampler,
optional shaker, and sample collection methods as provided in U.S.
Pat. No. 9,575,063 and US 2016/0097707, each of which is herein
incorporated by reference in its entirety, such rates can be
achieved together with automated sample feeding to the aspiration
needle. In addition, the flow cytometer is programmed with features
such as autocleaning of the aspiration needle to reduce likelihood
of sample carryover and cross-contamination.
[0043] In a preferred embodiment, the optical engine within the
flow cytometer is further characterized as having a set of lasers,
such as from single to multiple lasers (e.g., 2 or 3 or 4 or 5, or
even more), each tuned to a different wavelength suited for
excitation of fluorescent molecules. In some embodiments, improved
focusing of each of the plurality of laser beams to distinct
locations along the flow cell is accomplished by providing a set of
beam shaping optics for each laser, wherein each set preferably
includes two lenses to adjustably focus light horizontally along an
x-axis to a same horizontal position and vertically along a y-axis
to a different vertical position along a same excitation plane, the
plane being characterized as being within a flow path through a
flow cell of the flow cytometer. For the beam shaping optics
described here, the laser light propagation direction is defined as
Z-axis, which is normal to the horizontal x-axis and vertical
y-axis. Beam shaping optics preferably include cylindrical lenses
so that the focused beam is at the center line in the flow cell and
of elliptical shape. By assigning beam shaping optics to each
laser, each laser can be precisely focused to a different vertical
position of the flow cell thereby eliminating the tradeoffs
associated with configurations that require sharing beam shaping,
steering and guiding optics between lasers as commonly provided in
commercially available systems.
[0044] In related embodiments, multiple lasers share certain
beam-shaping optics components and at the same time, each
individual laser can be focused and steered or guided to different
vertical positions along a same plane. Those skilled in optics
design may develop such optical illumination systems with the
guidance herein.
[0045] In preferred embodiments, a set of optics is provided, which
includes collection optics that collect particle-scattered light
and fluorescence from the flow cell and filtration optics that
filter the collected fluorescence (collected by the collection
optics) emitted from the flow cell into different wavelength
ranges, wherein the set of optics further separate the fluorescence
of a same wavelength range into different locations according to
the lasers by which the fluorescent light is excited; and a
detector that detects light from each of different locations, each
excited by one individual laser, thereby distinguishing between
fluorescence emitted within the same wavelength range from
different lasers within the set of lasers and converts light to an
electrical signal.
[0046] It is important to note that the collection optics and
filtration optics not only collect particle-scattered light and
fluorescence from the flow cell but also filter the collected
fluorescence from the flow cell into different wavelength ranges.
Furthermore, the collection optics and filtration optics take
advantage of separated laser focal points along the flow cell (also
referred to as within the excitation plane) for different lasers,
allowing the separation of fluorescent signals of same wavelength
ranges as excited by the different lasers into different locations
where a detector is employed to detect and measure fluorescent
light excited by each different laser.
[0047] Fluorescence signals and scatter signals can be detected
with various optical detectors. Focusing the lasers at distinct
positions along the flow cell for excitation permits comparisons
between the timing of fluorescence signals at each detector at
different wavelengths and forward scatter signals, which help
construct the light signals (the scatter light, forward scatter and
side scatter, and the fluorescent signals at different wavelengths
and excited by different lasers) into data sets corresponding to
individual cells or particles going through the excitation plane in
the flow cell. Note that the data sets are digital signals that are
converted from analog electronic signals obtained through light
detectors that convert light into electronic signals.
[0048] For example, for a 3 laser system having red, blue and
violet lasers, the laser beams are focused to 3 different vertical
locations along the flow cell, ordered as red, then blue, then then
violet counting from a lower position to a higher position.
Consider a particle is labelled by a number of different
fluorescent dyes, where two fluorescent dyes are excited by violet
laser, emitting light at .about.450 nm and .about.780 nm, two
fluorescent dyes are excited by blue laser, emitting light at
.about.530 nm and .about.780 nm, and one fluorescent dye is excited
by red laser, emitting light at .about.780 nm range. As the
particle moves through these 3 laser beams, the fluorescent light
at .about.780 nm induced by red laser excitation would be ahead the
fluorescence at .about.780 nm and the fluorescence at .about.530 nm
induced by a blue laser, which is then followed by the fluorescent
light at .about.780 nm and at .about.450 nm induced by a violet
laser. Assuming that a forward scatter is detected for particles
passing through the blue laser beam, the timing of forward scatter
for a particle would coincide with the .about.780 nm and .about.530
nm fluorescence induced by blue laser. Similarly, assuming that a
side scatter is detected for particles passing through the blue
laser beam, the timing of side scatter for a particle would
coincide with the .about.780 nm and .about.530 nm fluorescence
induced by blue laser. Note that the collection optics would
collect the side scatter and all fluorescence at different
wavelength ranges (i.e. .about.450 nm, .about.530 nm, and
.about.780 nm range) excited by the three lasers. The filtration
optics would split the collected fluorescence into light with
wavelength around 450 nm, light with wavelength around 530 nm and
light with wavelength around 780 nm. Light detectors are then
placed to detect fluorescence at 450 nm and 530 nm respectively. In
addition, with collection optics and filtration optics, the
filtered light with wavelengths around 780 nm would be separated
into three different locations according to the laser by which the
fluorescence is excited. Then three different detectors can be
employed so that each is to detect the fluorescence (at .about.780
nm range) each excited by one laser. In one exemplary embodiment,
these different locations are separated by a couple mm to a few mm
apart within a focal plane of the collection optics (as the
fluorescence is being collected from the flow cell and, being
filtered and focused down onto a focal plane of the collection
optics). In another embodiment, the fluorescence separated by a few
mm could be further separated into even larger distances, for
example 5-10, or 10-20 mm apart.
[0049] In the above example, optical detectors can detect forward
scatter, side scatter, as well as fluorescence at 450 nm range
(excited by violet laser), fluorescence at 530 nm range (excited by
blue laser), and three different fluorescence at 780 nm range
excited by red laser, blue laser and violet laser, respectively.
Signal processing approaches and algorithms assign and combine the
signals obtained from each detector into data sets that belong to a
single cell or particle.
[0050] The flow cytometer is preferably provided as part of a flow
cytometry system, which includes computer software for acquiring
and analyzing flow cytometry data. The flow cytometry software
operably communicates the flow cytometer to a computer and provides
a variety of easy to use features. Among these include slideable
compensation scroll bars positioned adjacent to corresponding
fluorescent channels on displayed data plots, an easy to use
experiment manager, and improved laboratory reports showing gated
populations and corresponding counts.
[0051] A preferred flow cytometry system includes configurable
detection fluorescence channels; 1 to 5 or 6 lasers; optimized
detector conditions, automated fluid-maintenance functions; syringe
pump sampling fluidic system; novel optical design, with enhanced
signal detection as a powerful analytical tool for cell-by-cell
discrimination. This system permits reliable quantitative
measurements and rapid acquisition of statistically significant
data for high density, multiplexed assays.
[0052] Many of the improvements described herein have been
achieved, in part, are due to the adaptation of a multi-pixel
photon counter (MPPC) chip for detecting fluorescent light.
Compared to photomultipler tubes (PMTs) widely used in flow
cytometry, MPPC technology presents some distinguishing features,
such as a smaller foot print in size and larger quantum efficiency,
allowing the measurement of low light signals. MPPC, as also known,
silicon photomultiplier (SiPM), is a solid state device with an
array of avalanche photodiodes (APD) operated in the Geiger mode.
When operating in the Geiger mode, sufficiently large electrical
charge output is produced at each individual APD even when a single
photon excites it. Each pixel is a combination of an avalanche
photodiode (APD) operating in Geiger mode and a resistor (referred
to as a "quenching resistor"), where the APD is placed in series
with the resistor. In operation of using MPPC for light detection,
the light beam (fluorescence or scattered light) is directed to the
MPPC surface. MPPC output as a result of receiving the incident
light beam is in a form of electrical current, which is then
converted to an analog voltage signal. The analog voltage signals,
preferably, are converted to digital signals using an analog to
digital converter (ADC) and processed in digital form for increased
accuracy and speed. Whilst the digital output data is not directly
from the MPPC detector/device, for simplicity in the present
document and invention, we sometimes refer the digital data from
the ADC or from digital circuits after ADC as the MPPC output data
itself. It is worthwhile to point out the incident light beam to
MPPC surfaces may be a constant light beam, or in many applications
including flow cytometry applications, the incident light beam may
be of a pulse form, and as such, the MPPC output would also take
the form of a pulse, that is the output is time-dependent. Here the
MPPC output includes the time-dependent electric current from MPPC,
or the time-dependent analog voltage after current-to-voltage
conversion, or the time-dependent digital data after ADC.
[0053] Whilst MPPCs could theoretically be used in flow cytometry
to detect and measure fluorescent light, it has not been used in
practical cytometers, due to a number of technical challenges:
[0054] 1) MPPC has a large dark-count background. Such a large dark
count background presents a limiting factor for detecting dim
fluorescent signals. Furthermore, such dark count could be
temperature dependent. [0055] 2) MPPC gain, dependent on the
operational voltage applied to a MPPC, is very much temperature
dependent. Temperature fluctuation could result in a change in
light signal amplification gain. This is different from PMT where
temperature does not have a large influence on the PMT gain. [0056]
3) MPPC's linear dynamic range in terms detecting and measuring
light of different intensities is limited, as mainly dependent of
number of pixel in the MPPC. A wide dynamic range covering many
decades of light intensity is required for detectors usable for
flow cytometry applications.
[0057] Challenges associated with MPPC's temperature dependent,
large dark-count, have been overcome by effectively reducing and
controlling the dark-count noise by designing and developing an
apparatus to lower and/or stabilize the operational temperature of
MPPCs. By securing control of MPPC's operating temperature, the
MPPC's gain during operation has now been stabilized. Further an
un-expected `by-product` effect of lowering the operational
temperature of the MPPC is that is we have achieved a large dynamic
range since the low end light detection is mainly determined by the
dark-count.
[0058] Whilst MPPC's linear dynamic range is limited when using
conventional flow cytometry optics' configurations with
conventional light detectors (e.g. namely PMTs), we have developed
an approach to manipulate the beam size at the MPPC sensor surface
to maximize usage of MPPC light-detection area for all filter
channels and to provide sufficient dynamic range of light
detection.
[0059] As described above, various approaches commonly-used to
separate and split light could also be employed to separate, split
and collect the fluorescent light and the scattered light from the
particles flowing through the flow cell. Among these include
beam-shaping, steering, and guiding optical components.
[0060] Configuring a set of optics having multiple-lenses at
precise locations relative to each other and relative to flow cell
can be employed to collect the fluorescent light and the
diffraction light from the particles. The collected light is then
split into different channels according to the particular
excitation lasers and according to the light wavelength. The light
beam at each fluorescent channel due to one excitation laser and
with a particular wavelength range can be detected with a MPPC with
an appropriate beam size through the use of different optical
lenses for focusing or expanding the light beams.
[0061] In one approach of light collection and separation,
different fiber optics cables are used to collect the
fluorescent/scattered light as excited from different laser
sources. Then the light from each fiber optical cable is split into
different fluorescent channels via use of different dichroic
mirrors and bandpass filters. The light beam at each fluorescent
channel with a particular wavelength range can be detected with a
MPPC with an appropriate beam size through the use of different
optical lenses for focusing or expanding the light beams.
[0062] In another approach, a specially designed objective is used
to collect light from particles as they pass through different
laser sources and the light is separated into different beams
according to which laser source the light was generated. Each
separated light beam, originating from one laser source, is then
separated into different fluorescent channels according to the use
of different dichroic mirrors and bandpass filters. Similar to
above, the light at each fluorescent channel with a particular
wavelength range can be detected with a MPPC with an appropriate
beam size through the use of different optical lenses for focusing
or expanding the light at such channel.
[0063] In one embodiment of the present invention, the optical
engine comprises a set of optics, which includes collection optics
and filtration optics that separate fluorescence of a same
wavelength range into different locations in a focal plane of the
collection optics according to the lasers by which the fluorescent
light is excited. Preferably, the optical engine further comprises
a lens for expanding the beam size of fluorescence light of the
same wavelength range from each of different locations of the focal
plane of the collection optics, each originating from the
fluorescence excited by an individual laser, to about a size
between 1 mm to 3 mm, at the MPPC surface. Optionally, the beam
size at MPPC surface is between 1.5 and 2 mm. Whilst it is
desirable to utilize the largest beam size possible on a MPPC
surface, compromise may have to be developed for positioning
tolerance/accuracy. In addition, for increasing linearity dynamic
range of MPPC surface, a properly developed/designed lens would
allow a relatively-uniform beam be achieved on the MPPC
surface.
[0064] By developing optic lenses matched with the fluorescence
light previously separated by the different locations of the focal
plane of the collection optics, relatively-uniform beam with
suitable sizes can be achieved at chosen MPPC surfaces.
Surprisingly, such a beam uniformity (less than 10% variation in
the light intensity across the beam size) and such a beam size
(about 70% in single dimension, relative to a MPPC width/length,
e.g. 3 mm, or 6 mm) could be obtained for the fluorescence light of
all the wavelength ranges. i.e for all fluorescent channels for the
optical collection system and the properly designed optic lens
here. Together with such beam characteristics at MPPC surfaces,
appropriately applied operational voltages on MPPC (affecting the
gain and dark-count of MPPC) and suitably controlled temperature
range (affecting the dark count), a reasonable linear dynamic range
can be observed. For a number of commercial available MPPCs, we
have been able to obtain a linear range from about 3 decades, about
4 decades, to about 5 decades, under a single operational voltage,
for different MPPCs. Such a surprisingly positive result of an MPPC
providing about 3 decades, about 4 decades, or above linear dynamic
range, as described here, can be achieved only through the
development of associated optics allowing suitable beam size,
uniform beam distribution, controlled dark counts etc. To be clear,
in the present invention, each decade corresponds to a factor of
ten, thus 3, 4, and 5 decades correspond to a factor of 1,000,
10,000 and 100,000, respectively. In other words, a linear range of
3, 4 and 5 decade means that the MPPC output goes linearly with the
intensity of the incident light for the linear dynamic response
range where the ratio of its maximum to its minimum light intensity
value is 1,000, 10,000 and 100,000, respectively.
[0065] To further improve the linear dynamic range of an MPPC,
additional technological approaches are required. As discussed
above, MPPC's linear dynamic range in terms detecting and measuring
light of different intensities (i.e. the outputting-electronic
current as a result of incident light) is mainly dependent of
number of pixels in the MPPC. Specifically, a given pixel within an
MPPC can be activated by an incident photon (with a probability
less than 1 for such activation, this probability is the Quantum
efficiency for the MPPC detector), resulting a nano-second range
response pulse in the output electronic current. At basic physics
level, such an activation in a MPPC pixel is the status where the
avalanche photodiode (APD in this pixel) operates in Geiger mode
under an operational bias voltage, in series with a quenching
resistor. During such nano-second range response time (dependent on
pixel capacitance and quench resistance), an additional photon
arriving at the same pixel will not be able to activate the pixel.
Thus, in theory and in practice, at any given instant or within a
short time window of nano-seconds range, the maximum number of
pixels for an MPPC that can be activated (that are being activated)
by the incident photons for electronic current output is the number
of pixel within the MPPC, and any more photons (more than the
number of pixels for the MPPC) arriving at MPPC surfaces will not
be able to activate more pixels and will not contribute to
additional electronic current output. This is the main cause of the
limitation for MPPC's linear dynamic range of electrical current
output in relationship to the input light (at MPPC surface). Some
additional factors, other than pixel numbers of a MPPC detector as
well as MPPC's quantum efficiency for photon detection, that could
influence the dynamic ranges, include the dark count (or dark
current), the MPPC gain (the ratio of electrical charge of the
response pulse generated from one activated pixel due to one
incident photon, divided by the charger per electron), the
cross-talk factor (the cross talk refers to an effect where an
activated pixel that detects a photon for output charge pulses may
affect other pixel, causing them to produce output electric charge
pulse). At the experimental level, an operational bias voltage on
MPPC would affect all above factors, including quantum efficiency,
the dark current, the gain as well as crosstalk factor.
[0066] To further improve the dynamic range of a MPPC so that the
output current is linearly proportional to the light intensity over
a wide intensity range, a numerical calibration technique has been
developed in the present invention. The technique is based on the
fact that at relatively high level of incident light intensity to
MPPC surfaces, the output current from MPPC may saturate and would
be lower than the ideal situation where all the incident photons
could generate an output electric charge pulse. If the ratio of the
actual output current from an MPPC to the theoretically-ideal
output current can be determined at each and all light intensity
for these high light level situations, these ratios could be used
to calibrate the MPPC detector output by dividing the measured MPPC
output data by such ratios. We term these ratios `calibration
factors`.
[0067] The approach to derive the calibration factors has been
developed, as following. For a given MPPC type, a number of
standard light beams are designed and produced through choice and
designs of optical source (e.g., laser, laser diode, light emitting
diode), beam shaping optics (e.g. spherical, aspherical,
cylindrical lenses), light intensity attenuation mechanism (e.g.
neutral density filters that have a constant attenuation across the
range of visible wavelengths, beam cutting pinholes, as well as
control voltages on the light source that may modulate the output
intensity levels of light source), the light wavelength range (e.g.
Band pass filters, light sources of given wavelength ranges), the
light pulse shape or waveform. These standard light beams could be
reliably and consistently produced having the desired beam size and
beam uniformity by using the appropriately designed beam shaping
optics. The beam quality parameters including beam size and beam
distribution uniformity should match, or be the same as, those of
the fluorescence or scattering light to be detected in the optical
engine or the flow cytometer of the present invention. The standard
light beams can have different wavelength ranges, for example,
515-545 nm, 650-670 nm, etc, which should match the wavelength
ranges used for optical detection (scattering or fluorescence) in
the optical engine and the flow cytometer. The light intensity of
the standard beam can be adjusted, having a range of intensity from
sub-milli-watts, to micro-watts, to nano-watts or pico-watts, or
even femto-watts ranges, which should match the light intensity
range that will be detected by an MPPC in the optical engine or the
flow cytometer of the present invention. The light pulse shape or
waveform is designed and controlled so that they would match the
light pulse waveform that is detected at an MPPC in the optical
engine or the flow cytometer of the present invention.
[0068] The light intensities of such a number of standard light
beams are measured and determined using certain optical detectors
having a good wide dynamic range and an excellent linearity
response. Such optical detectors may be chosen from a PMT (photon
multiplier tube) and an APD (avalanche photon diode). These optical
detectors may not have sufficient detection sensitivity or
linearity for accurately measuring low light intensity beams. To
ensure an accurate determination of light intensities of all
standard light beams covering a wide intensity range, the low
intensity-level beams could be produced by one or multiple light
attenuation filters (e.g. neutral density filter having Optical
Density (OD) 0.5 or OD 1, optical density is the negative of the
common logarithm of the transmission coefficient) and such beam
intensities could then be calculated based on the OD numbers of the
filters as well as the measured light beam intensity before going
through OD filters. Thus, we have been able to produce a range of
standard beams with quantified light intensity levels. In other
words, for a range of target light intensity levels, we can operate
light sources, change optic paths and adjust various possible
conditions (e.g. voltage applied to light source, or adding or
removing certain light attenuation filters) to produce light beams
(of desired size, uniformity, wavelength) to meet these target
intensities.
[0069] With the capability for reliably producing such a number of
standard light beams, MPPC of interest is used to measure these
standard beams at a given operational bias voltage for the MPPC.
MPPC, together with its associated circuits, including
current-to-voltage conversion, as well as analog-to-digital
convertor (ADC) and any possible analog or digital filters, will
provide a series of output digital data, each, corresponding to a
light intensity level of a standard light beam. Thus, a plot of
MPPC digital output versus the light intensity levels could be
obtained for such a number of standard light beams. At the
intensity range of low light levels but still a few times above
dark-count of the MPPC detector, an excellent linearity can be
obtained (observed, as expected) between the MPPC output data and
light-intensity level. Based on the slope (the measured MPPC data
is in Y-axis, the light intensity is in the x-axis, a slope can be
derived based on linear regression for a number of data points) in
this good linearity range, it is possible to obtain theoretical
MPPC output data for all the light intensity beams as the product
of the slope (in the above linearity range) and the light intensity
values for each standard beam.
[0070] Thus, a calibration factor for each measured MPPC value can
be obtained by dividing the measured MPPC data by theoretical MPPC
data for each of standard light beams. Mathematical modelling or
simulation can be then undertaken to derive a calibration factor
equation so that for any measured MPPC data, a calibration factor
is determined and used to calculate the `theoretical, calibrated`
MPPC output data. As such, the non-linearity of MPPC output versus
input-light intensity can be corrected or calibrated so that an
extended wide-dynamic range is possible.
[0071] In above paragraphs, we have described the process to derive
calibration factors for calibrating the MPPC output data based on
use of a number of standard light beams with a range of
intensities. The above approach is not intending for limiting the
technique or methods for deriving or utilizing such calibration
factors for extending the dynamic range of an MPPC detector.
Indeed, there are other possible methods or approaches to derive
the calibration factors. Regardless the approaches, the calibration
factors, being the ratio between the measured, linearity-limiting
MPPC values and the ideal, theoretical MPPC values that are
expected from an ideal, linearly-responding MPPC.
[0072] It is worthwhile to point out that the calibration factors
or calibration factor curves would be dependent on MPPC
types--different MPPC types would have different calibration factor
curves. Calibration factor curves are also dependent on operational
bias voltages applied to an MPPC and operational temperature.
Furthermore, the calibration curves depend on the size and
uniformity distribution of light beams on the MPPC surfaces, as
well as on the wavelength ranges of the light beam.
[0073] In preferred embodiments of the optical engine employing
MPPC detectors, each MPPC output data is scaled up by dividing the
data with a corresponding calibration factor, for the purposes of
improving linear dynamic range of the MPPC detectors. The
calibration factors are determined using the techniques described
above. Preferably, the calibration factors allow the improvement of
linear dynamic range by at least about half (0.5) decade. More
preferably, the calibration factors allow the improvement of the
linear dynamic range by at least about one (1) decade. Even more
preferably, the calibration factors allow the improvement of the
linear dynamic range by at least about one-and-half (1.5) decade.
Even more preferably, the calibration factors allow the improvement
of the linear dynamic range by at least about two (2) decades.
[0074] In view of the above, the invention is described in still
more detail with reference to the following non-limiting
embodiments. Turning first to FIG. 1, a pump 16 drives sheath fluid
to a flow cell 130, into which a sample is also delivered by other
known mechanisms such as a pump (e.g. a syringe pump). The sample,
conventionally embodied as a suspension of particles (e.g. cells),
is hydrodynamically focused by the sheath fluid into the center of
the flow cell. The ordered passage of cells through different
excitation lasers generates fluorescent light, which is detected by
detectors following light collection and light splitting optics,
collectively referred to as a set of optics. There are various
methods and approaches for driving and delivering sheath fluid and
sample fluids containing suspensions of cells into a flow cell for
hydrodynamic focusing of sample fluid. These are well known in the
flow cytometry arts and are typically accomplished using a
combination of pumps 16, valves 18 and flow passages 19.
[0075] The flow cytometer 10 can be operated manually such as by
individually providing a suspension of cells tube-by-tube to the
flow cytometer 10, through aspiration via a sample aspiration
needle 14 (FIG. 1) as known in the flow cytometry arts, or may be
adapted for high throughput through the incorporation of an
optional modular autosampler. Such a modular autosampler is
preferably compatible with different loading tubes. Among these
include racks of conventional 12.times.75 mm flow tubes, 1.5/2.0 mL
tubes, such as those commonly manufactured by Eppendorf
International, multi-well plates, such as 24 well, 96 well, (even
348 well), flat bottom, V-bottom, round bottom or any other
suitable tube or dish for maintaining a suspension of cells. In
preferred embodiments, the autosampler is equipped with a shaker
for sample agitation or other mechanisms for sample mixing. In
preferred embodiments the autosampler includes self-alignment
protocols for ease of setup and maintenance, allowing convenient
installation by users.
[0076] In preferred embodiments the flow cytometer 10 includes a
microprocessor (i.e. one or multiple microprocessors) to control a
variety of functions, such as fluid or sample delivery, cell
suspension or shaking, and self-alignment of sample vessels. The
microprocessor is typically provided on a circuit board, coupled to
memory and electrically connected to electric mechanisms such as
electric pumps and actuators to accomplish the intended function.
Further, the microprocessor may modulate voltages to the detectors,
lasers, aspirating pump or other electrical components. The
microprocessor may include an analog to digital converter for
converting analog signals from the photodetectors to digital
signals. The microprocessor may process and analyze the digital
signals for different scatter channels and different fluorescent
channels to produce a data set for individual cells or particles.
The microprocessor is communicatively connected to a computer,
which provides various control commands to the microprocessor and
receives the data from the microprocessor, controlled by the
developed software.
[0077] Preferred embodiments include executable control programs
stored in the microprocessor for automated sample aspiration needle
14 cleaning (when a clean command is received from the computer
connected to the cytometer) after every sample aspiration to reduce
risk of cross contamination of cell suspensions. This is still more
preferred when using an autosampler. This is accomplished by
controlling various pumps and valves for cleaning external and
internal surfaces of the aspiration needle(s) 14 with sheath fluid
or rinse fluid. Preferably such a feature should result in less
than 1%, or 0.5%, even more preferably 0.1% or 0.05% carry over in
embodiments using such an autosampler.
[0078] Preferred embodiments also include an automated de-bubble
and unclogging feature, which prevents erroneous results from
bubbles or clogs in the fluidic flow of cell suspensions and
further ensures accurate direct absolute counts without needs of
expensive reference counting beads. In preferred embodiments, the
flow cytometer 10 also includes an automated cleaning function at
start up and shut down of the flow cytometer 10. This programming
improves the ease of use and removes the need of the user to
perform these steps, which can be tedious and time consuming. In
still further embodiments an automatic fluid level detection alarm,
such as in the form of a suitable fluid-level sensor is
incorporated to inform the user when system fluid levels are
low.
[0079] Turning to FIG. 2, a schematic providing an overview of an
exemplary optical engine 100 is shown. The optical engine 100
includes from one to three lasers 110, and a set of beam shaping
optics 120 for each laser 110 to independently shape and guide each
excitation light source to the flow cell 130. Fluorescence is
collected by collection optics 150, and separated into different
wavelength ranges by filtration optics 140. Detection of
fluorescence is accomplished using a MPPC detector 160 for each
channel. Many electronic components including one or more
microprocessors for operation of the flow cytometer 100 including
automated system functions in response to commands from the
developed control software, electronic circuits for operating light
detectors and for converting analog to digital signals and for
processing the digital signals etc. are not shown in FIG. 2 for
simplicity. The syringe pump fluidics in fluidic connection with
flow cell 130 during operation is also not shown here.
[0080] Although the optical engine 100 may use a single laser 110,
the optical engine 100 permits the flow cytometer 10 to perform
multicolor flow cytometry analysis such as by measuring a large
number of parameters and a number of fluorescence signals from each
fluorescently labeled cell. This can be achieved with, for example,
three lasers 110v, 110b, 110r in FIG. 2. Conveniently, lasers 110
can be added, removed, or interchanged at least in part due to the
individually assigned collection optics 150. An exemplary
configuration for multi-color flow cytometry is shown in FIG. 2,
including a first laser 110v emitting a wavelength of 405 nm (also
referred to as violet laser), a second laser 110b emitting a
wavelength of 488 nm (also referred to as blue laser), and third
laser 110r emitting a wavelength of 640 nm (also referred to as a
red laser). The skilled artisan will appreciate that the
interchangeability of one or more lasers 110 permits the user to
begin with a base flow cytometer system and add additional or
different lasers 110 as experiments dictate such need. It is
possible that during such interchange or exchange of one or more
lasers 100, the corresponding beam shaping optics 120 may be
changed or adjusted to achieve the optimal delivery of the laser
beam at the center of the flow cell 130.
[0081] FIG. 3 provides a simplified schematic of a top view showing
beam shaping optics 120 for three exemplary optical paths (P1, P2,
P3) of three laser beams from the three lasers (e.g. 110v, 110b,
110r), where a first cylindrical lens 112v for the first laser 110v
(e.g. 405 nm) focuses excitation light along an X-axis and a second
cylindrical lens 114v focuses the excitation light from the first
laser 110v along the Y-axis. Similarly, a first cylindrical lens
112b for a second laser 114b (e.g. 488 nm), focuses the excitation
light along an X-axis and a second cylindrical lens 114b focuses
the excitation light along a Y-axis. A set of two beam expanding
lenses 111r expand excitation light from the third laser 110r (e.g.
640), followed by a first cylindrical lens 112r focusing the
excitation light along an X-axis and a second cylindrical lens 114r
for focusing the excitation light along a Y-axis. The light paths
P1, P2, P3 from each laser 110 are focused to a single flow cell
130, through which cells are hydrodynamically focused into a narrow
stream in the central region of a flowing sheath fluid.
[0082] Also shown is a half-ball lens 152 for collecting
fluorescent light and side scatter light emitted from fluorescently
labeled cells while travelling through the flow cell 130. An
obscuration bar 180 is also shown, which blocks a raw laser beam
passing from P2 through the flow cell 130 and towards the forward
scatter (FSC) focusing lens 182, which itself focuses FSC light
from the second laser 110b (e.g. 488 nm) through a band-pass filter
184 (488/10 nm) for detection by a photodiode 186, which receives
the FSC light and converts it to an electrical signal for data
acquisition.
[0083] FIG. 4 is an enlarged schematic showing the three light
paths (P1, P2, P3) directed through the corresponding second lens
114v, 114b, 114r converging at a common/same focusing position H (a
centerline) along the flow cell 130 but focusing the laser beams at
different vertical positions Vv, Vb, Vr along the flow cell 130. As
a nonlimiting example, differential vertical focusing can be
accomplished by adjusting the second cylindrical lens 114v, 114b,
114r to direct one beam above and one beam below a center beam. For
example, the configuration shown in FIG. 3 vertically focuses Vv
the first light path P1 from the first laser 110v upwards, by a
given distance, (e.g. 80 .mu.m) relative to the vertical focusing
positioning Vb of the light path P2 via the second cylindrical lens
114b from the second laser 110b. This results in vertical focusing
Vv of the first laser beam/light path P1 in the flow cell 130
vertically above the vertical focusing Vb of the second light path
P2 by a same distance, (e.g. 80 .mu.m). Similarly, adjusting the
second cylindrical lens 114r for the third laser 110r downwards by
a given distance (e.g. 80 .mu.m) relative to the vertical position
Vb of the second cylindrical lens 114b for the second laser 110b
results in the vertical focusing Vr of the third laser beam P3 in
the flow cell 130 vertically lower by the same distance (e.g. 80
.mu.m) relative to the second laser beam P2. The skilled artisan
will appreciate that while this separation is depicted 80 .mu.m
apart, separations more or less than these could be performed. In
some embodiments the beams P1-P3 are separated from neighboring
beams P1-P3 by 70 .mu.m. In other embodiments, separation is 50
.mu.m, 55 .mu.m, 60 .mu.m, 65 .mu.m, 75 .mu.m, 85 .mu.m, 90 .mu.m,
100 .mu.m, 125 .mu.m, 150 .mu.m, 175 .mu.m, or 200 .mu.m.
Furthermore, adjusting the Z-axis positions of the three
above-mentioned second cylindrical lenses 114v, 114b, 114r allows
the three laser beam's Z-axis focal points coincide with the center
line in the flow cell 130, thus leading to the coincidence of laser
beams' Z-axis focal points within the focused sample narrow stream.
By focusing at different vertical positions Vv, Vb, Vr along a flow
cell 130, the fluorescence signals emitted by a particle traveling
through three focused laser beams at different vertical positions
is then collected by collection optics, filtered to different
wavelength ranges according to filtration optics, and can be
further separated into different locations on a same plane within a
couple of mm to a few mm distances between neighboring beams
according to laser sources by which fluorescence is excited.
[0084] Referring collectively to FIGS. 1-4, there are unique
advantages for independently controlling three separate optical
paths P1, P2, P3 from three lasers 110v, 110b, 110r. First, it is
straightforward to separately adjust the alignment of each focused
beam along the X-axis to the hydrodynamically-focused sample
stream, by adjusting the X-axis position of first cylindrical lens
112. This can be used to eliminate interference between different
laser beams and between different optical paths P1, P2, P3, thus
allowing each laser beam to be adjusted to optimal alignment.
[0085] Secondly, one can independently adjust the Z-axis positions
of the second cylindrical lens 114 for each laser beam, ensuring
the coincidence or alignment of the focal plane for the Y-axis beam
with the hydrodynamically-focused fluid stream in the flow cell
130. Again, there is no interference between different laser beams
and between different optical paths P1, P2, P3, thus allowing each
laser beam to be adjusted to the optimal alignment with the
fluidics.
[0086] Thirdly, one can readily adjust and control the separation
distance along the Y-axis between three different beams in the flow
cell 130, by simply adjusting and moving the height of the second
cylindrical lens 114 along the Y-axis for each laser beam. With
such an approach, one could adjust the beam separation distance
continuously over a relatively-large range.
[0087] Fourthly, by providing separate beam shaping and beam
guidance optics for each laser independently of the others, one
could choose or use lasers 110 having the same or different raw
beam diameters, since for each channel different cylindrical lenses
112, 114 could be used with different focal lengths to accommodate
the difference in the raw beam diameters.
[0088] Fifthly, such an optical illumination design would allow
easy configuration and possible upgrade of the laser illumination
sources. For example, one could start with a system having a single
laser 110 as excitation source. When there is a need for providing
additional laser 110 sources of different wavelengths, the new
laser(s) 110, together with its (their) corresponding beam shaping
and guidance optical components 120, could be readily added to the
system, without affecting the existing laser 110 and its beam
shaping optics 120. These unique advantages would allow optimal
alignment and focus of each laser beam in the flow cell 130,
overcoming the limitations compared to other light illumination
designs where different lasers share the same optical path for beam
shaping and beam separation, and focus and alignment for different
lasers need to be compromised.
[0089] Forward light scatter (also referred to as forward scatter
or low angle scatter) refers to the measurement in flow cytometry
that involves light refracted forward due to the passing of a cell
through a laser beam and is roughly proportional to the diameter of
the cell. When no cell is passing through the path of the laser
beam the beam passes uninterrupted through the flow cell 130 and is
blocked by an obscuration bar 180 so that no light from the laser
beam itself would arrive in the detector. However, when a cell
passes through the flow cell 130, light is refracted in all
directions. The light refracted in the forward direction misses the
obscuration bar 180 to reach the forward scatter detector 186.
[0090] In U.S. Pat. No. 9,575,063, herein incorporated by reference
in its entirety, we described the development of novel light
obscuration bar. To this end, the methods, devices and systems
herein preferably include a light obscuration bar 180, such as
those described in U.S. Pat. No. 9,575,063. In a preferred
embodiment, a diamond shaped obscuration bar 180 is provided. In
another embodiment an obscuration bar 180 that is of a rectangular
shape and has its horizontal dimension being the same as or longer
than its vertical dimension is provided for blocking the incident
laser beam. In still another embodiment, the perimeter of the
obscuration bar 180 follows a contour of a light intensity
distribution plot for blocking incident laser beam. In a still
further embodiment, the obscuration bar 180 follows a contour of a
light intensity distribution plot within the 0.1% contour line. A
0.1% contour line or boundary corresponds to a line where the light
intensity at each point on the contour is at 0.1% of maximum light
intensity of the incident light. An obscuration bar 180 following
the contour of a light intensity distribution plot within the 0.1%
contour was determined to block 99% of the unscattered beam from
the FSC detector. Accordingly, the invention also provides an
obscuration bar 180 generally diamond shaped that follows a contour
of a light intensity distribution plot within the 0.1%, 0.2%. 0.5%,
1.0% or 2.0% contour line and methods of its shaping.
[0091] Flow cytometry conventionally includes the labeling of cells
with one or more fluorophores. This is typically performed by
adding a fluorescently labeled reagent, such as a fluorescently
labeled antibody against a surface marker, to a suspension of
cells, then washing away unbound reagent. Fluorophores present on
or in the cell as it passes through the laser beam adds to the
cumulative signal from the cell. Such reagents are well known in
the art and available from many suppliers. Both side scatter and
fluorescence are collected through collection optics 150 positioned
generally orthogonal to the laser beam path then filtered and
reflected into different channels using filtration optics 140, such
as dichroic mirrors. Dichroic mirrors permit passage of a certain
wavelength ranges and reflect the remaining wavelengths.
[0092] In the context of the present invention, the flow cytometer
10 incorporates an optical engine 100, including: a set of lasers
110, each emitting light at a specific wavelength suited for
excitation of fluorescent molecules; where lights from lasers are
focused horizontally along an x-axis to a same horizontal position
H and focused vertically along a y-axis to a different vertical
position Vv, Vb, Vr along a same excitation plane, wherein the
plane is characterized as a flow path through a flow cell 130 of
the flow cytometer 10; a set of optics, which includes collection
optics 150 for collecting particle-scattered light and fluorescence
from the flow cell, and filtration optics 140 that filter the
collected fluorescence from the flow cell 130 into different
wavelength ranges, wherein the set optics further separate the
fluorescence of a same wavelength range into different locations in
a focal plane of the collection optics according to the different
lasers 110 by which the fluorescent light is excited; and a
detector 160 that selectively detects light at the different
locations, thereby distinguishing between fluorescence emitted
within the same wavelength range from different lasers 110v, 110b
and 110r within the set of lasers 110 and converts light to an
electrical signal. The preferred embodiment of optical detectors
160 in the present invention are embodied as MPPC detectors 160.
Each fluorescent or measurement channel among a plurality of
channels can therefore be characterized as belonging to a
wavelength range emitted using a single laser 110v, 110b, 110r from
the set of lasers 110. To this end, a plurality of channels provide
a plurality of data sets for sample analysis.
[0093] A more detailed overview is shown in FIG. 5, where
fluorescence and SSC signals from a flow cell 130 are collected
using collection optics 150 then split into different detection
channels with filtering optics 140, primarily comprised of long
pass and/or short pass dichroic mirrors 142 and bandpass filters
144. Shown are a number of MPPC modules (161a-161h) for the
detection of the following fluorescent wavelength ranges: 725/40 nm
(161a), 697/58 nm (161b), 661/20 nm (161c), 780/60 nm (161d),
615/20 nm (161e), 586/20 nm (161f), 445/45 (161g), 530/43 (161h).
Each MPPC module 161a-h comprises at least one MPPC detector 160,
for converting optical light signal to electronic signals. The MPPC
detectors 160 are further electrically connected to circuitry (not
shown for simplicity) for analog to digital conversion, and the
converted digital signals are then be processed by microprocessors
that are in communication with a computer (also not shown).
Further, TABLE 1 provides a nonlimiting listing of compatible
fluorophores for conducting 21 color (or 21 channel) flow cytometry
analysis, when each MPPC detector could be used to derive
fluorescence signal from different lasers. To this end, each
channel within the 21 channel flow cytometry system is
characterized by a particular originating laser and a wavelength
range.
TABLE-US-00001 TABLE 1 Excitation 445/45 530/43 586/20 615/20
661/20 697/58 725/40 780/60 Pacific Blue 405 nm X BV 421 405 nm X
DAPI 405 nm X BV510 405 nm X AmCyan 405 nm X PACIFIC 405 nm X
ORANGE BV605 405 nm X QDOT 605 405 nm X BV650 405 nm X QDOT 655 405
nm X BV711 X QDOT 705 405 nm X BV786 X QDOT 800 405 nm X
Fluoroscein 488 nm X FITC 488 nm X ALEXA FLUOR 488 nm X 488 GFP 488
nm X PE 488 nm X PE-CY 5 488 nm X PERCP 488 nm X 7-AAD 488 nm X
PERCP-CY5.5 X PERCP- 488 nm X eFluor710 PE 561 nm X PE-Texas Red
561 nm X PE-Cy5 561 nm X PE-Cy5.5 561 nm X PE-Cy7 561 nm X APC 640
nm X ALEXA FLUOR 640 nm X 680 ALEXA FLUOR 640 nm X 700 APC-CY 7 640
nm X
[0094] By focusing excitation laser beams at distinct vertical
positions along the flow cell 130 and collecting light from the
flow cell 130, the light from each of the vertical positions is
collected and split into different wavelength ranges, such as shown
in FIG. 5. Furthermore, the light from each of the vertical
positions, when collected and filtered down through the filtration
optics, was separated to about a couple millimeters to a few
millimeters between neighboring beams, according to the laser
source by which the fluorescence light is excited.
[0095] A more detailed schematic of the collection and measurement
of spatially distinct positions in a flow cell is shown in FIG. 6A.
In this exemplary embodiment, fluorescence from four distinct
vertical positions (Vv, Vr, Vg, Vb) along a flow cell 130 is
collected using a high refractive index half ball lens 152 (for
example, n is about 1.5 or above). The optical path of each
proceeds through two sets of doublet lenses 154 (with a large
NA>0.8 or above) for collimating fluorescent light and filtered
through one or more dichroic mirrors 142. As already eluded to, the
half-ball lens 152 is preferably made of high refractive index
materials. Such a choice is for maximize the Numerical Aperture
(N.A.) and for reducing the overall diameter/size of the
fluorescent light beam following the half-ball lens 152. This
reduced beam diameter also permits a smaller diameter for the
doublet lens 154, making the design and manufacturing of such a
doublet lens 154 somewhat less-difficult. It has been found that
the combination of high refractive index half-ball lens 152 and
doublet lenses 154 results in a high numerical-aperture objective
for collecting fluorescent light efficiently. Such a design not
only results in a high collection efficiency but also leads to a
reduced cost for design and manufacturing of such objectives. The
optical path then proceeds through a bandpass filter 144, and to an
MPPC module 161.
[0096] As illustrated in FIG. 6A, light from each of the distinct
vertical positions Vv, Vr, Vg, Vb is focused to a small beam size
(for example, about a couple of millimeters or less) with a
separation distance of a couple of millimeters to a few millimeters
between neighboring beams as light is collected through collection
optics (halfball lens 152, two sets of doublet lenses 154) and
travels through the filtration optics (dichroic mirror 142,
bandpass filter 144), reaching the MPPC detection module 161. At a
focal plane 200 of the collection optics (halfball lens 152, two
sets of doublet lenses 154), adjacent beams of a same wavelength
range are spaced a couple to a few millimeters apart. To be clear,
in this case, the collection optics comprising halfball lens 152
and two sets of doublet lenses 154 collects scattered light and
fluorescent light from different locations (Vv, Vr, Vg, Vb) of the
follow cell and focus the light onto a focal plane 200 where each
focused beam size is, generally, less than 500 microns in diameter.
For the example shown in FIG. 6A, the MMPC module 161 includes a
prism mirror 168 that further separates apart the light from the
two middle vertical positions in the flow cell 130 so that each
light beam is detected independently through a MPPC detector 162,
163, 164, 165. Note in one particular embodiment, the focal plane
200 coincides with the mirror-reflection points of the prism mirror
168. In FIG. 6A, Total 4 detectors 162, 163, 164 and 165 are used
to detect fluorescence light from the four distinct vertical
positions Vv, Vr, Vg, Vb in the flow cell 130, as excited each
individual laser.
[0097] In a preferred embodiment, each light beam goes through a
lens for expanding the beam before reaching an MPPC detector. This
is shown in FIG. 6B. The light beams from four different vertical
positions Vv, Vr, Vg, Vb in the flow cell 130 travel through
different optical components within the set of optics before
reaching MPPC detector surfaces in the following manner. Similar to
FIG. 6A, the light from the two middle vertical positions Vr, Vg in
the flow cell 130 is further separated apart by the prism mirror
168. The light reflected by the upper surface of the prism 168
reaches the reflection mirror 172, then goes through a lens 193
before reaching the surface of MPPC detector 163 for detection.
Similarly, the light reflected by the lower surface of the prism
168 reaches the reflection mirror 174, then goes through a lens 195
before reaching the surface of MPPC detector 165. The light from
the top vertical positions Vv in the flow cell 130 goes through a
lens 192 and reaches the surface of MPPC detector 162 for
detection. The light from the lowest vertical positions Vb in the
flow cell 130 goes through a lens 194 and reaches the surface of
MPPC detector 164 for detection. The use of the lenses 192, 193,
194 and 195 expands the beams to an improved size for detection at
MPPC surfaces (a consistent and optimized beam size is possible
through the design and the use of the lenses 192, 193, 194 and
195). In addition, the lenses 192, 193, 194 and 195 serve an
additional design function of resulting a uniform beam distribution
at MPPC surfaces. Such an optimized beam size and beam uniformity
(through the design of the lenses 192, 193, 194 and 194) helps the
improvement of linear-dynamic detection range of MPPC devices being
used for light detection here.
[0098] An additional important feature of the present invention is
a multi-pixel photon counter (MPPC) chip to detect fluorescent
light. Comparing with PMTs widely used in flow cytometry, MPPC
presents some distinguishing features such as smaller foot print in
size and larger quantum efficiency, allowing the measurement of low
light signals. Whilst MPPCs could theoretically be used in flow
cytometry to detect and measure fluorescent light, it has not been
used in practical cytometers, for a number of reasons such as a
large dark-count background, the gain being temperature dependent,
and possibly limited dynamic range.
[0099] To this end, in another embodiment of the present invention,
an optical engine for use in a bench top flow cytometer is
provided, which includes, a laser, tuned to a wavelength suited for
excitation of fluorescent molecules, wherein light from the laser
is focused horizontally along an x-axis to a horizontal position
and vertically along a y-axis to a vertical position along an
excitation plane, wherein the horizontal position along the
excitation plane intersects a flow path through a flow cell of a
flow cytometer; a set of optics comprising collection optics for
collecting fluorescence emitted from the flow cell and filtration
optics that filter the collected fluorescence from the flow cell
into different wavelength ranges, thereby providing different
fluorescent channels; and an MPPC detector at each fluorescent
channel to detect fluorescence and convert light to an electrical
signal. In a preferred embodiment, the optical engine further
comprises a set of lasers, wherein each of the lasers is focused
vertically along the y-axis to a different vertical position along
the same excitation plane, further wherein the set of optics
separate the emitted fluorescence from the flow cell into different
fluorescence channels, wherein each channel is characterized by a
different wavelength range and a different laser by which the
fluorescence is excited.
[0100] In preferred embodiments of above optical engines, the MPPC
is operated with a linear dynamic range above 3 decade. More
preferably, the MPPC is operated with a linear dynamic range above
4 decade.
[0101] In some embodiments of above optical engines, the MPPC
digital output value is corrected according calibration factors.
Preferably, the calibration factors improve linear dynamic range of
the MPPC by more than half decade. More preferably, the calibration
factors improve linear dynamic range of the MPPC by more than one
decade. Even more preferably, the calibration factors improve
linear dynamic range of the MPPC by more than one and one-half
decade. Still, even more preferably, the calibration factors
improve linear dynamic range of the MPPC by more than two
decades.
[0102] In a preferred embodiment, forward scatter (FSC)
characterization of cells includes a FSC detector, a FSC focusing
lens to collect FSC light, and an obscuration bar that blocks an
incident laser beam from entering the FSC focusing lens and the FSC
detector. The relationship between timing of fluorescence signal at
a fluorescent light detector and timing of forward scatter signal
at a FSC detector provides an approach for determining which laser
induces excitation of a detected fluorescent signal in a detection
channel.
[0103] Further improvement of forward scatter (FSC) detection has
been achieved through the use of improved obscuration bars. In a
preferred embodiment, a diamond shaped obscuration bar is provided.
In another embodiment an obscuration bar that is of a rectangular
shape and has its horizontal dimension being the same as or longer
than its vertical dimension is provided for blocking the incident
laser beam. In still another embodiment, the perimeter of the
obscuration bar follows a contour of a light intensity distribution
plot for blocking incident laser beam. In a still further
embodiment, the obscuration bar follows a contour of a light
intensity distribution plot within the 0.1% contour line. A 0.1%
contour line or boundary corresponds to a line where the light
intensity at each point on the contour is at 0.1% of maximum light
intensity of the incident light. An obscuration bar following the
contour of a light intensity distribution plot within the 0.1%
contour was determined to block 99% of the unscattered beam from
the FSC detector. Accordingly, the invention also provides an
obscuration bar generally diamond shaped that follows a contour of
a light intensity distribution plot within the 0.1%, 0.2%. 0.5%,
1.0% or 2.0% contour line and methods of its shaping.
[0104] Components of the optical engine are preferably housed as a
single unit, and some of these optical components can be removed
and interchanged for modification with other components. To this
end, a housing configured to house optical engine components is
also provided. The housing includes the optical engine components
such as the set of lasers, the optics for focusing laser beams to
the excitation plane, collection optics, filtration optics,
photo-detectors or light-detectors, further filters (and/or
lenses), as well as an electrical interface for electrical
connection from the photo-detectors or light-detectors to
electrical circuitry, which would be connected to an external
microprocessor or a remote computer. In some embodiments, each
laser has a corresponding set of beam-shaping optics wherein light
from each laser is focused horizontally along an x-axis to a same
horizontal position and vertically along a y-axis to a different
vertical position along a same excitation plane, wherein the same
horizontal position along the excitation plane intersects a flow
path through a flow cell of a flow cytometer. In other embodiments,
all the laser beams being independently adjustable horizontally
along an x-axis and independently adjustable vertically along a
y-axis, are combined together via suitably placed dichroic mirrors
and go through a common achromatic beam shaping optics so that all
the laser beams are focused to a same horizontal position and to
different vertical positions along a same excitation plane, wherein
the horizontal position on the excitation plane interests a flow
path through a flow cell of the flow cytometer. Preferably, the
components within a same housing are configured for
interchangeability of different lasers, focusing lenses, long pass
and short pass dichroic mirrors, filters, pinhole passages and
detectors. This is accomplished by standardizing engagement
features such as positioning of alignment holes, snaps, screws or
other fasteners across different components for interchangeability
and by providing a set of beam shaping optics for each laser
individually. Preferably, the photo-detectors or the light
detectors are MPPC detectors. In some embodiments, a flow channel
is mounted in the housing and configured for coupling to a flow
cytometer apparatus for hydrodynamic focusing of samples including
particles (e.g. beads or cells) by tubing connectors.
[0105] In furtherance of the above embodiments, FIG. 7A shows that
the dependency of the digital output after converting electronic
analog signals from an MPPC on the power level of the incident
light. For this test, the incident light was pulsated at 10 kHz
with a pulse duration of 4 .mu.s. The analog electrical current
output from MMPC chip is converted to a voltage with a resistor.
The voltage is then digitized to give digital output. The incident
light beam was varied from about 115 um to 1.1 mm. Clearly, the
larger the beam size, the wider the linear dynamic range. For this
MPPC chip of about 1.5 mm.times.1.5 mm in size, out of the beam
sizes being evaluated shown in FIG. 7A, 1.1 mm beam size gave the
better dynamic ranges than those for beam sizes of 760 um, 550 um
and 115 um. The beam size refers to the diameter of light spot for
the incident beam on the MPPC surface.
[0106] FIG. 7B shows that the dependency of the digital output from
another MPPC detector with different resistors from that used in
FIG. 7A. Here the resistors are used for converting MPPC output
electric current to an analog electrical voltage, which is further
converted to a digital MPPC output with an ADC. The resistors in
FIG. 7B are larger than those used in FIG. 7A, as such, the digital
output from FIG. 7B is larger than that in FIG. 7A. Similar to the
data in FIG. 7A, 1.1 mm beam size gave better dynamic ranges than
those for beam sizes of 760 um, 550 um and 115 um. FIG. 7C shows a
linear regression fit of MPPC digital output versus incident light
intensity for the beam size of 1.1 mm. As evident in FIG. 7C, the
MPPC output for the incident beam size shows a linear dynamic range
about 3.5 decade. That is to say, for the light intensity between
about 10.sup.-11 W to about 3.times.10.sup.-8 W, the MPPC shows a
linear response in terms of the digital output being proportional
to the light intensity.
[0107] FIG. 8 shows that the dependency (diamond symbol on broken
line) of the digital output after converting electronic analog
voltage signals from an MPPC on the power level of the incident
light. For this test, the incident light was pulsated at 10 kHz
with a pulse duration of 4 .mu.s. The analog electrical current
output from MMPC chip is converted to a voltage with a resistor.
The voltage is then digitized to give digital output. The incident
light beam was 1.9 mm for this MPPC chip of about 3 mm.times.3 mm
in size. The beam size refers to the diameter of light spot for the
incident beam on the MPPC surface. The solid line in FIG. 8 shows a
linear regression fit of MPPC digital output versus incident light
intensity for the measured MPPC data versus light intensity. As
evident in FIG. 8, the MPPC output for the incident beam size shows
a linear dynamic range above 4 decade. That is to say, for the
light intensity between about 2.times.10.sup.-11 W to about
3.5.times.10.sup.-7 W, the MPPC shows a linear response in terms of
the digital output being proportional to the light intensity.
[0108] FIG. 9 shows the plot of calibration factor versus MPPC
digital output, as determined for an MPPC having size of 3
mm.times.3 mm in size, at a particular operational bias voltage and
room temperature, for incident light beam of wavelength range
between 515-545 nm. Each calibration factor was determined using
the technique described in the above sections of the present
invention. Continuous line shows a theoretical modelling of the
dependency of the calibration factor on the MPPC digital output
using a polynomial equation. This derived equation allows the
calculation of calibration factor for any MPPC digital output
value. By dividing the MPPC digital output values by corresponding
calibration factors, the MPPC output values are corrected and the
resulting output data would show significantly improved linearity.
The linear dynamic range for the MPPC data showing in FIG. 9 would
be improved by more than 1.5 decade when the calibration factors
are used to calibrate or correct MPPC digital output values.
[0109] The lower end of the MPPC dynamic range, when the incident
light is about a few pW or less, is limited by the optical noises
and electronic noises of the system, as well as, very importantly,
the dark count of MPPC. In this regard, we have been reducing and
controlling the dark-count noise by lowering the operational
temperature of MPPCs. FIG. 10 shows that the histogram of dark
count noises with MPPC blocked from any external light with left
panel at a room temperature and the right panel at a lowered
temperature. In this test, MPPC is powered at certain operational
voltages. The output `dark-count` noises in electrical current were
converted into a voltage through a resistor. The output voltage is
then digitized through an analog-to-digital convertor. The digital
output is then processed through algorithm identifying a max value
within each small time interval during entire experiment process.
This way, each time interval would lead one output value from MPPC
circuit. The output values are then plotted in a histogram format.
Clearly, the larger the mean for the histogram, the larger the dark
count noises. As the temperature for the MPPC chip was reduced,
there was a significant reduction in the mean of the distribution
of MPPC dark-count output from a digital value of 247 to 70, an
about 70% reduction.
[0110] Thus, we have shown that reducing the MPPC temperature could
reduce the dark count noises. We are now developing special
techniques and approaches to lower down the operational temperature
of the MPPC.
[0111] FIG. 11 shows a preliminary data of 6-pk beads detected at
the fluorescent channel of 530/43 nm, excite by a blue laser, by a
MPPC detector, with left panel at a room temperature and the right
panel at a lowered temperature. In this test, MPPC is powered at
certain operational voltages. The 6-pk beads were running through
the flow channel in a flow cell, where laser beams of 488 nm, 640
nm and 405 nm were focused into a same horizontal position and each
to a different vertical position separated by 80 um in the center
of the flow cell. The fluorescence generated by the beads as they
were excited by laser beam were collected through a collection
optics and filtered into a wavelength range of about 530 nm/43 nm.
The fluorescence due to the blue laser was then detected with a
MPPC chip. Clearly, at room temperature showing on the left panel,
the detector can barely distinguish two dimmest beads. Yet with
reducing the temperature on the right panel, the MPPC detector can
readily resolve the two dimmest populations. This clearly
illustrates that reducing the operation temperature could improve
the system resolution and dynamic range of the fluorescence
detection.
[0112] Control and communication to the flow cytometer for setup
and data acquisition is performed using a computer communicatively
coupled to the flow cytometer. Accordingly, flow cytometry software
for loading in a computer has also been developed. The flow
cytometry software preferably includes data acquisition features
and data analysis features. As known in the flow cytometry arts,
data acquisition involves the collection and storing of data from
an experiment. This may also include set up features for acquiring
data, such as compensation adjustment, defining the number of cells
to be counted within a particular gated population, setting a
sample flow rate, and the other experiment controls encountered in
the flow cytometry arts. Data analysis features may include
plotting cell subpopulations across one or more fluorescent colors,
determining absolute counts for particular cell subpopulations,
determining relative percentages of particular subpopulations, cell
cycle analysis, as well as other data analysis features found in
flow cytometry programs. To this end, the software provides
versatile, user friendly and intuitive plotting and gating tools
and its statistical tools provide exceptional statistical data
analysis capabilities. In preferred embodiments, all acquisition
parameters, experiment and sample files, along with plots are
visible and accessible in one window area.
[0113] In some embodiments, a data collection process can include
calculating and processing fluorescence signals of a same
wavelength for different detectors into different fluorescence
channels (as excited by different lasers, at different vertical
positions of an excitation plane); generating a graphical user
interface (GUI) that displays two-dimensional plots with one
parameter versus another one (e.g. FSC vs. SSC, FSC vs. a
fluorescence signal, or one fluorescence signal vs. another
fluorescent channel), wherein the GUI further has compensation
scroll bars adjacent to the comparison plots to adjust compensation
of spectral overlap between one or more channel; collecting data
from each light scatter channel and from each fluorescence channel;
and saving the data into a data file. The software also preferably
includes a gating function that permits the user to select a
subpopulation from one of the histogram plots or one of the 2-D
plots and generate another plot (which could either be a
one-parameter histogram plot or be a two-parameter 2-D plots for
the selected subpopulation. This "gating" and further analysis of
"gated populations" can be repeated.
[0114] In still another related embodiment a flow cytometry method
is provided, which includes providing flow cytometer or flow
cytometer system as provided herein; labeling a sample of cells
with a plurality of fluorescent labels; pumping the sample of cells
through the flow channel; collecting flow cytometry data; and
analyzing the flow cytometry data to determine the presence,
absence or abundance of one or more of the plurality of fluorescent
labels on or in cells of the sample.
* * * * *